title: Mechanistic basis of excitation-contraction coupling in human pluripotent stem cell-derived ventricular cardiomyocytes revealed by Ca2+ spark characteristics: direct evidence of functional Ca2+-induced Ca2+ release.
abstract: BACKGROUND
  Human embryonic stem cells (hESCs) serve as a potential unlimited ex vivo source of cardiomyocytes for disease modeling, cardiotoxicity screening, drug discovery, and cell-based therapies. Despite the fundamental importance of Ca(2+)-induced Ca(2+) release in excitation-contraction coupling, the mechanistic basis of Ca(2+) handling of hESC-derived ventricular cardiomyocytes (VCMs) remains elusive.
  
  
  OBJECTIVE
  To study Ca(2+) sparks as unitary events of Ca(2+) handling for mechanistic insights.
  
  
  METHODS
  To avoid ambiguities owing to the heterogeneous nature, we experimented with hESC-VCMs, purified on the basis of zeocin resistance and signature ventricular action potential after LV-MLC2v-tdTomato-T2A-Zeo transduction.
  
  
  RESULTS
  Ca(2+) sparks that were sensitive to inhibitors of sarco/endoplasmic reticulum Ca(2+)-ATPase (thapsigargin and cyclopiazonic acid) and ryanodine receptor (RyR; ryanodine, tetracaine) but not inositol trisphosphate receptors (xestospongin C and 2-aminoethyl diphenylborinate) could be recorded. In a permeabilization model, we further showed that RyRs could be sensitized by Ca(2+). Increasing external Ca(2+) dramatically escalated the basal Ca(2+) and spark frequency. Furthermore, RyR-mediated Ca(2+) release sensitized nearby RyRs, leading to compound Ca(2+) sparks. Depolarization or L-type Ca(2+) channel agonist (FPL 64176 and Bay K8644) pretreatment induced an extracellular Ca(2+)-dependent cytosolic Ca(2+) increase and reduced the sarcoplasmic reticulum content. By contrast, removal of external Na(+) or the addition of the Na(+)-Ca(2+) exchanger inhibitor (KB-R7943 and SN-6) had no effect, suggesting that the Na(+)-Ca(2+) exchanger is not involved in triggering sparks. Inhibition of mitochondrial Ca(2+) uptake by carbonyl cyanide m-chlorophenyl hydrazone promoted Ca(2+) waves.
  
  
  CONCLUSIONS
  Taken collectively, our findings provide the first lines of direct evidence that hESC-VCMs have functional Ca(2+)-induced Ca(2+) release. However, the sarcoplasmic reticulum is leaky and without a mature terminating mechanism in early development.
authors.affiliation: Stem Cell and Regenerative Medicine Consortium; Department of Physiology, LKS Faculty of Medicine, University of Hong Kong, Hong Kong.
authors.forename: Sen
authors.initials: S
authors.lastname: Li
authors.affiliation: Institute of Molecular Medicine, Peking University, Beijing, People's Republic of China.
authors.forename: Heping
authors.initials: H
authors.lastname: Cheng
authors.affiliation: Division of Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, Maryland.
authors.forename: Gordon F
authors.initials: GF
authors.lastname: Tomaselli
authors.affiliation: Stem Cell and Regenerative Medicine Consortium; Department of Physiology, LKS Faculty of Medicine, University of Hong Kong, Hong Kong; Center of Cardiovascular Research, Icahn School of Medicine at Mount Sinai, New York, New York. Electronic address: ronaldli@hku.hk.
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Li
mesh_terms: D001145:Arrhythmias, Cardiac / Q000378:metabolism* / Q000473:pathology; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002478:Cells, Cultured; D056966:Excitation Contraction Coupling; D006352:Heart Ventricles / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
chemical_list: D020746:Calcium Channels, L-Type; D002118:Calcium
keywords: AP; CCCP; CICR; CM; Ca(2+) sparks; Ca(2+) waves; Ca(2+)-induced Ca(2+) release; EC; FDHM; FWHM; I(Ca,L); IP3R; L-type Ca(2+) current; MLC2v; NCX; Na(+)-Ca(2+) exchanger; RyR; SERCA; SR; Stem cell; VCM; Ventricular cardiomyocytes; action potential; carbonyl cyanide m-chlorophenyl hydrazone; cardiomyocyte; excitation-contraction; full duration at half maximum; full width at half maximum; hESC; human embryonic stem cell; iPSC; induced pluripotent stem cell; inositol trisphosphate receptor; myosin light chain 2v; ryanodine receptor; sarco/endoplasmic reticulum Ca(2+)-ATPase; sarcoplasmic reticulum; ventricular cardiomyocyte